Dermalife k lifefactors kit

The hydrogel monomers, 4-arm PEG4norb, were purchased from JenKem Technology USA (Plano, TX, USA). The photoinitiator LAP was synthetized as described previously [52 ]. The fluorescent dye Alexa Fluor488 was obtained from Invitrogen (Waltham, MA, USA), and propidium iodide from Carl Roth GmbH (Karlsruhe, Germany). Trypsinized bovine collagen type I, PureCol, was obtained from Advanced BioMatrix (Carlsbad, CA, USA), and fibronectin from Corning GmbH (Wiesbaden, Germany). Purified water was filtered with MilliQ system, Merck Millipore, Burlington, MA, USA. TGFα, insulin and BPE (bovine pituitary extract) were supplied as parts of the DermaLife K LifeFactors kit from Lifeline Cell Technology (Frederick, MD, USA). Human recombinant EGF (E. coli-derived) was obtained from PromoCell (Heidelberg, Germany), and recombinant human TGFβ-1 (CHO-derived) was purchased from Peprotech (Hamburg, Germany). EGF receptor monoclonal antibody 225 was obtained from Life Technologies (Carlsbad, CA, USA). All other reagents; i.e., PEG-dithiol; FITC-dextran, 40 kDa; 10xDMEM; 7.5% NaHCO₃; fluorescein diacetate; mitomycin C; and EGFR inhibitor AG-1478, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

HT-1080 fibrosarcoma cells (DSMZ, Braunschweig, Germany) were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; both Sigma-Aldrich, St. Louis, MO, USA). Normal human epithelial keratinocytes (nHEK; CellSystems, Troisdorf, Germany) were cultured in DermaLife basal medium supplemented with DermaLife K LifeFactors kit (both Lifeline Cell Technology, Frederick, MD, USA), and Penicillin/Streptomycin antibiotics (Gibco, Waltham, MA, USA). The final component concentrations in the supplemented (complete) DermaLife medium were 5 μg/ml insulin, 6 mM L-glutamine, 1 μM epinephrine, 5 μg/ml apo-transferrin, 100 ng/ml hydrocortisone hemisuccinate, 0.4% bovine pituitary extract, 100 U/ml Penicillin, and 100 μg/ml Streptomycin. Only nHEK cells up to passage number 5 were used. The cultures were maintained at 37° C and in 5% CO₂ humidified atmosphere.